{"paper_id":"d630ed9f-9869-4281-aeb3-524c07b9e936","body_text":"Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women ( 1 ). Menstrual dysfunction,\nanovulation, hyperandrogenism, hirsutism, and polycystic ovaries are considered as the symptoms of PCOS ( 2 ).\nHyperandrogenism stimulates follicular atresia in granulosa cells via apoptosis. As a result, apoptosis and oxidative stress can interfere with follicular growth in women\nReceived: 23/August/2019, Accepted: 05/January/2020\n*Corresponding Address: P.O.Box: 1449614525, Department of Anatomical\nSciences, School of Medicine, Iran university of Medical Sciences, Tehran,\nIran\nEmail: Shirazi.r@iums.ac.ir\nRoyan Institute\nInternational Journal of Fertility and Sterility\nVol 14, No 2, July-September 2020, Pages: 143-149\nsuffering from PCOS ( 3 ). Therefore, disruption of follicular growth in patients suffering from PCOS is related\nto granulosa cells apoptosis and oxidative stress caused\nby high production of reactive oxygen species (ROS).\nSince mitochondria can regulate apoptosis and ROS production, these organelles may be affected by high rates\nof follicular atresia in the PCOS patients ( 4 ). Changes in\nthe mitochondrial function might cause insulin resistance, oxidative stress, hyperandrogenism, and glucose intolerance, leading to the appearance of PCOS symptoms ( 5 - 7 ).\nThe disturbance of mitochondrial function in granulosa\ncells may cause some disorders in oocyte function, maturation, and fertilization. This may also affect the fertility\nof PCOS patients by reducing the oocyte quality. Therefore, the proper functionality of mitochondria is of importance in this regard ( 5 ). Mitochondria play a key role\nin the determination of numerous factors involved in the\nreproduction, such as oocyte quality, follicular growth,\ndevelopment, and granulosa cell proliferation ( 6 ). Mitochondria also mediate various cellular processes, including apoptosis, ROS production, calcium signaling, adenosine triphosphate (ATP) synthesis, pyrimidine synthesis,\nand Fe-S protein synthesis ( 3 ,  8 ). Furthermore, mitochondria, as the main organelle for cellular ROS production,\ncan also impair mitochondrial DNA ( mtDNA ), which may\nsubsequently be the cause of different diseases. Being\nmore susceptible to oxidative damage and attaining high\nrates of mutations are more common in the mitochondrial\ngenome than nuclear DNA due to the adjacency to the\nelectron transport chain (ETC), lack of sheltering histones\nand inefficient DNA repair capabilities ( 9 ). mtDNA of\nmammals is nearly 16 kb in size, encrypting 13 proteins of\nthe oxidative phosphorylation (OXPHOS) complexes, 22\nribosomal RNAs ( rRNA ), and transfer RNAs ( tRNA ) that\nare required for mitochondrial mRNA translation.  mtDNA , like nuclear DNA, can influence mitochondrial gene\nexpression, biogenesis, and function through epigenetic\nmodifications ( 10 ). It is of note to mention that mitochondrial biogenesis can also influence mtDNA and nuclearencoded protein synthesis, the congregation of the double\ngenetic origin derived proteins,  mtDNA  replication as well\nas cell growth and proliferation ( 11 ). Mitochondrial biogenesis is hard to understand and needs several processes,\nsuch as synthesis of  mtDNA  and nuclear genes ( 12 ). The\nmain gene in mitochondrial biogenesis that is critical for\n mtDNA  transcription and maintenance is mitochondrial\ntranscription factor A ( TFAM ). Mitochondrial biogenesis\nis also regulated by nuclear genes such as NRF2, which\ncontrols the other factors in mitochondria ( 11 ).\nSince oocyte quality is a crucial factor for conception in\nPCOS patients and that depends on mitochondrial function and structure, prescription of an appropriate medication may improve fertility rate as a consequence of improved oocyte quality ( 13 ). Different treatments have been\ntrialed, and vitamin D3 is one of which to have shown\nsigns of improvement in PCOS patients ( 6 ). Vitamin D3\nhas been used before to alleviate signs of insulin resistance, hyperandrogenism, and oxidative stress in PCOS\npatients and other metabolic disorders ( 14 ). Vitamin D3\nalso has an important role in calcium homeostasis, cellular proliferation, and differentiation( 15 ). Recently it has\nbeen demonstrated that the low level of vitamin D can\nresult in excessive androgen secretion, insulin resistance,\nand follicular growth interruption in the patients suffering from PCOS. These occur through the decline of sex\nhormone-binding globulin (SHBG) levels, insulin receptors, and calcium dysregulation ( 8 ). The serum concentration of 25-hydroxyvitamin D in women with PCOS is less\nthan 20 ng/ml, which can exacerbate PCOS symptoms\n( 16 ). Therefore, in this study, we aimed to investigate the\neffects of vitamin D3 on the mitochondrial biogenesis,\nmembrane integrity, and  mtDNA  copy numbers in the\ngranulosa cells isolated from PCOS-induced mice.\n\nThis is an experimental study that the effect of vitamin\nD3 on mitochondrial biogenesis in a PCOS mouse model\nwas investigated. Androgen excess and other symptoms\nof PCOS were induced by the injection of DHEA (Sigma,\nAustria), 6 mg/100 g body weight. DHEA was dissolved\nin 95% ethanol (0.01 mL) and mixed with sesame oil (0.09\nmL). Subsequently, it was injected subcutaneously into\nfemale BALB/C mice (25 days old) for 20 consecutive\ndays before reaching puberty (PCOS group, n=20). As a\nvehicle control, 0.1 mL of sesame oil (Sigma, Austria) and\n0.01 mL of 95% ethanol (Sigma, Austria) were injected\ninto another group of the same mouse strain for 20 consecutive days (n=20). A Control group of the same mouse\nstrain without any treatment was also considered (n=20).\nThe mice were kept at room temperature (25 ± 1°C, RT),\nwith enough food and water, and under diurnal modulation by daily light. All the animal trials were performed\nin agreement with the Institutional Animal Care Committee of Iran University of Medical Sciences and Health\nServices for animal welfare. (ethics code: IR.IUMS.REC\n1396.29969). The weight changes in mice were measured\nevery day. Vaginal smears were also taken every day over\nthe 20-day course of treatment. The mice were sacrificed\nby cervical dislocation. For histological assessments, the\novaries were subsequently fixed with 10% formalin (Merck, Germany). Next, 5-μm sections were made with a microtome, and the sections were immersed in xylene and\nethanol with different grades for deparaffinization and\nrehydration, respectively (Merck, Germany). The ovaries\nwere then stained with hematoxylin and eosin (DAKO,\nUSA). For morphology assessment, the ovaries assessed\nby a Nikon microscope (Nikon, Japan), and photographs\nwere taken.\nFor the analysis of sex hormones, cardiac blood\nsamples were collected using needles. Blood serum\nwas subsequently separated using a centrifuge machine\nat (300 rpm, 4°C, 10 minutes) and follicle-stimulating\nhormone (FSH), luteinizing hormone (LH), 17β-estradiol\n(E2) and progesterone levels were measured by an\nELISA kit (Abcam, Cambridge, UK) according to the\nmanufacturer's guidelines.\nThe ovaries of 45-day BALB/C mice (DHEA-reated and the vehicle group) were removed after the mice were\nsacrificed via cervical dislocation. For aspiration of the\nfollicles, 25-gauge needles were used, and the follicles\nwere aspirated in a solution made of phosphate buffer saline (PBS) and 1.0% bovine serum albumin (BSA) (Invitrogen, USA). 70-μm cell strainers (BD Falcon, MA,\nUSA) were used to isolate granulosa cells from the other\ncells and tissues. Subsequently, granulosa cells were separated from the oocytes with a 40-μm cell strainer (BD\nFalcon, MA, USA). Blood cell contamination was removed by RBC lysis buffer after centrifugation at 1000\nrpm (4°C, 10 minutes). Then, the pellet was mixed with\nphenol red-free DMEM/F12 medium containing 10% fetal bovine serum (Sigma, Austria). The medium was centrifuged at 1000 rpm (4°C, 10 minutes). Next, the pellet\nwas removed and transferred to cell culture dishes containing DMEM-F12, 10% FBS (Sigma, Austria), 100 mg/\nmL streptomycin (Sigma, Austria), 100 IU/mL penicillin\n(Sigma, Austria), 2 mM glutamine (Sigma, Austria), 1\nmM sodium pyruvate (Sigma, Austria). The culture dishes were then incubated at 37°C, with 5% CO2 and 95%\nhumidity.\nTo identify granulosa cells, an antibody against FSHR,\na specific marker of granulosa cells, was used. Affinitypurified rabbit anti-follicle stimulating hormone receptor (FSHR) polyclonal antibody was purchased from\nantibodies-online (ABIN1872743). First, the cells were\nspread on a slide using a cytospin centrifugation device,\nand the slides were then immersed in a cold normal buffered formalin (NBF) solution to be fixed. Subsequently,\nthe cells were washed with PBS and blocked using PBSTriton/BSA. Afterward, the primary FSHR antibody was\nadded to granulosa cells overnight. The following morning the cells were washed three times with PBS and were\nsubsequently treated with the secondary FSHR antibody\nfor 30 minutes. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Preparations were washed\nin PBS before mounting on glass slides. Slides were\nviewed on an epifluorescence microscope and captured\nwith a digital camera.\nThe treatment groups for granulosa cells were as follows:\nPCOS granulosa cells treated with vitamin D3 (100\nnM) for 24 hours ( 17 ,  18 )\nPCOS granulosa cells without any treatments\nNon-PCOS granulosa cells (control group)\nThe Trizol reagent (Sigma, Austria) was used to extract\nthe total RNA of granulosa cells in all groups. Then, chloroform was added to the mixture of granulosa cells and the\nTrizol reagent. Afterward, the mixture was centrifugated\nat 1000 rpm (4 C, 10 minutes). The upper phase containing the total RNA was collected. Next, the total RNA was\nwashed with 75% ethanol, allowed to air dry, and then reconstituted in diethylpyrocarbonate (DEPC) water. Using\na cDNA synthesis kit (Thermo Scientific, USA), the total\nRNA was reverse-transcribed according to the manufacturer’s guideline. In summary, a mixture of the random\nhexamer, first-strand buffer (all from Fermentas), DNase-\n(Fermentas Inc, MD, USA) treated RNA, RiboLockTM\nRNase inhibitor, dNTP Mix, Dithiothreitol (0.1M) and\nSuperScriptTM II Reverse Transcriptase was made for\nreverse transcription of each sample. The thermocycler\n(company) was set at 25°C for 10 minutes, 43°C for 40\nminutes, and 75°C for 15 minutes. Quantitative PCR was\nperformed using 1 μl of cDNA in a reaction consisting of\nROXTM Reference Dye, SYBR Premix EX TaqTM (Takara, Japan), and 1 μl of the desired primer. The β-actin\ngene was utilized as a housekeeping gene. The reactions\nwere amplified with StepOne™ Real- Time PCR System\n(Applied Biosystems, MA, USA) as following: denaturation at 95 °C for 10 seconds, 35 cycles of amplification\n(95°C for 5 seconds and 60°C for 30 seconds), separation\nstage at 95°C for 15 seconds, 60°C for 1 minutes, and\n95°C for 15 seconds. Using oligo 7.60 software to design\nprimers. The TFAM forward primer was CCG AGC TCC\nTCC TCC TTT GC and the TFAM reverse primer was\nCCT ACA ACG CAG CGA CCG AG.\nFor the measurement of the  mtDNA  copy number, quantitative polymerase-chain-reaction (qRT-PCR) was used.\nForward primer and reverse primer were used to analyze\n mtDNA . SYBR Green I Master Mix (10 μl) (Sigma, Austria), which contains 10 pmol of reverse primer and 10\npmol of forward primer was mixed with DNA (10 ng).\nThe qPCR set-up consisted of 4 segments: 50°C for 2\nminutes, 95°C for 10 seconds followed by 40 cycles of\ndenaturation at 95°C for 5 seconds, annealing at 59°C\nfor 35 seconds, and extension at 72°C for 1 minutes. For\neach qPCR reaction, the copy number of the  mtDNA  and\nthe threshold cycle number (Ct) of the β-actin gene were\nmeasured. The runs were replicated at least two times, and\nthe normalization was performed against the housekeeping gene,\nβ-actin. For the quantification of the  mtDNA \ncopy number, the double delta Ct analysis was applied.\nIsolated granulosa cells were fixed using 2.5% glutaraldehyde in PBS and then treated with 1.0% osmium tetroxide in the same buffer for the post-fixation procedure.\nFor performing the dehydration process, ethanol and propylene oxide were used. Then inserted in epoxy resin, and\nsectioned. Using ethanolic uranyl acetate to contrast the\nsections and lead citrate and observed under a transmission electron microscope (Zeiss LEO 906 (TEM), 100 kV,\nGermany).\nThe data in this experiment are expressed as the means\nand standard error of the mean ( 19 ) for three independent biological replicates. Statistical significance between\ndifferent groups was evaluated and analyzed by on-way\nanalysis of variance (ANOVA), followed by Tukey’s post\nhoc test. The level of statistical significance was set at\nP<0.05. The SPSS software (version 21.0) was utilized\nfor the data analysis.\n\nIn the PCOS group induced by DHEA, the serum level\nof estradiol and LH were higher when compared to control and vehicle groups, respectively ( Table 1 ). The lower\namount of FSH serum was detected in the PCOS group\nversus the control group and vehicle group due to estradiol negative feedback. The ratio of LH/FSH was significantly increased in the PCOS group in comparison to the\nvehicle group ( Table 1 ). The estrous cycle was irregular in\nthe PCOS group and ultimately stopped, whereas, in the\ncontrol and vehicle group, normal cycles (nearly 5-7 days)\ncontinued as normal. Using mice in the control and vehicle\ngroups that were just in the estrous cycle to exclude the\ninfluence of the estrous cycles on other measurements.\nHormonal levels\nData are presented as mean ± SD. *; P<0.05, **; P<0.005, FSH; Follicle-stimulating\nhormone, LH; Luteinizing hormone, DHEA; dehydroepiandrosterone.\nUpon H&E staining, the specimen was analyzed under the light microscope. Normal follicles were detected at\nvarious developmental stages in the vehicle group. Corpus luteum was also observed in the control group, which\nwas an indicator of normal ovulation ( Fig .1A ). Due to the\nseizure of the estrus cycle, no corpus luteum was detected\nin the PCOS group ( Fig .1B ).\nHistological assessment of ovaries. A. Follicles of normal ovaries\nrepresented follicles at different stages, and corpus luteum (CL) and B.\nOvaries of the polycystic ovary syndrome (PCOS) model induced by dehydroepiandrosterone (DHEA) revealed antral and pre-antral follicles and\nsome cysts (FC) were observed in H&E staining. No corpus luteum was\nobserved in the PCOS ovary .Scale bar: 50 μm.\nTo assure that the cells being experimented on granulosa cells, a granulosa cell antibody was used against\nFSHR. Photograph analysis showed that the target cells\nwere stained with this antibody, indicating that they were\ngranulosa cells ( Fig .2 ). Thus, for the treatment of granulosa cells, vitamin D3 (100 nM) was used for 24 hours.\nSubsequently, RNA was extracted, and Reverse transcription- polymerase chain reaction (RT-PCR) performed to\nmeasure the expression of mitochondrial biogenesis gene\n(TFAM) in different groups. Vitamin D3 increased the expression of TFAM in the PCOS group ( Fig .3 ) by 5-fold\ncompared to the PCOS group without any vitamin D3\ntreatment. This indicates that vitamin D3 might stimulate\nmitochondrial biogenesis in PCOS-induced granulosa\ncells.\nFollicle-stimulating hormone receptor (FSHR) (specific markers of granulosa cells) was investigated. The FSHR expression in isolated granulosa cells\n(green) was observed. Nuclei (blue) were stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 100 μm.\nThe expression of TFAM (mitochondrial biogenesis gene) in cultured\ngranulosa cells of DHEA-induced PCOS BALB/C mice was compared between\nthree groups. Granulosa cells were pre-incubated in the serumfree medium in\nthe presence or absence of vitamin D3. The expression of the mitochondrial\nbiogenesis gene was upregulated in the vitamin D3 group. It is also revealed\nthat the gene expression was declined in PCOS granulosa cells in comparison\nwith non-PCOS healthy granulosa cells (control group), ***; P<0.05, DHEA; Dehydroepiandrosterone, and PCOS; Polycystic ovary syndrome.\nFor the analysis of the  mtDNA , qPCR was performed.\nOur results revealed that in the PCOS group treated with\nvitamin D3, the  mtDNA  copy number increased significantly in comparison to the non-treated PCOS group\n( Fig .4 ). Data analysis by the quartile distribution of  mtDNA \ncopy number in the non-treated PCOS group showed an\nassociation between  mtDNA  copy number and PCOS risk.\nThe mitochondrial DNA copy number (mtDNA) in cultured granulosa cells of DHEA-induced PCOS BALB/C mice was compared between\nthree groups. Granulosa cells were pre-incubated in the serum-free medium in the presence or absence of vitamin D3. The mitochondrial DNA\ncopy number was significantly increased in the vitamin D3 group in comparison with the non-treated PCOS group (**; P<0.05). It is also revealed\nthat the mitochondrial DNA copy number was declined in the non-treated\nPCOS granulosa cells in comparison with the non-PCOS healthy granulosa\ncells (control group). DHEA; Dehydroepiandrosterone, PCOS; Polycystic\novary syndrome.\nFor the evaluation of the alterations of the mitochondria structure, transmission electron microscopy was\nemployed. Most of the mitochondria in the PCOS group\nwithout any treatment were spherical, with almost no cristae; however, in the PCOS group treated with vitamin D3\nas well as in the non- PCOS group (control group) intact inner and outer membrane and a clear intermembrane\nspace was observed ( Fig .5 ).\nMitochondria membrane structure (TEM).  A.  PCOS group without\nany treatments were spherical with almost no cristae.  B.  PCOS group\ntreated with vitamin D3 and also in the non-PCOS group (control group)\ninclude undamaged mitochondria.\n\nThe present study demonstrated that vitamin D3 affected  mtDNA  copy number, mitochondrial structure, and\nmitochondrial biogenesis in granulosa cells of a PCOSinduced mouse model in comparison with healthy normal\novaries. PCOS is regularly described by oligomenorrhea,\nchronic anovulation, hyperandrogenism, and hyperinsulinemia ( 20 ). Androgenic hormones, such as DHEA, testosterone, and androstenedione, cause some problems in\nthe patients suffering from PCOS ( 2 ). According to previous studies, in the PCOS ovaries, the atretic follicles\nincreased that caused by hyperandrogenism, which is\ncritical in the pathogenesis of PCOS. High levels of androgen in women that suffer from PCOS might intensify\nfollicular atresia and follicular development disruption\nthat might cause subfertility ( 4 ). According to our previous study ( 17 ), to mimic the hyperandrogenism condition,\nfor induction of the PCOS model and also confirmation of\nthe abnormal hormonal level and ovarian morphological features in PCOS mice, DHEA was injected into the 25-\nday old female mice intraperitoneally. Androgen excess,\ninsulin resistance, and disturbed follicular development\nare some symptoms of this disorder that might interfere\nwith female fertility ( 21 - 23 ). The levels of LH, estradiol,\nalong with the ratio of LH to FSH were increased in the\nPCOS-induced mice compared to the vehicle group. The\nlevel of FSH was comparatively decreased in the PCOS\ngroup caused by the estradiol level feedback.\nDisrupted ovulation and oocyte quality induced by hyperandrogenism can be improved by different treatments\nsuch as metformin and spironolactone ( 7 ). Besides hormonal treatments, various supplements such as vitamin\nD3 have been shown to improve the PCOS symptoms\n( 24 ). Moreover, several lines of evidence demonstrate\nthe positive role of vitamin D3 in some disorders, such as\npremature ovarian failure (POF), endometriosis, PCOS,\nand male infertility ( 17 ,  25 ). It has been demonstrated that\nvitamin D3 might stimulate follicular development in patients with PCOS; however, it could not alleviate disrupted lipid and glucose metabolism ( 26 - 28 ). A large body of\nstudies has shown that vitamin D3 has constructive effects on alleviating the symptoms of ovulation disorders\nand insulin resistance in women suffering from PCOS\ndisorder ( 17 ,  29 ). Hormonal fluctuations in PCOS women\ncan be improved by vitamin D3; however, the duration of\ntreatment can influence the degree of symptom alleviation\n( 14 ,  26 ). Underlying mechanisms as to how vitamin D3\nexerts its effects are yet to be elucidated.\nFor the assessment of the effect of vitamin D3 on mitochondrial biogenesis, isolated granulosa cells from PCOS\novaries were treated with vitamin D3. Mitochondrial membrane integrity and alteration in mtDNA copy numbers were\nalso evaluated. It has been demonstrated that mitochondria,\nas the powerhouse of the cell, are of importance for optimum oocyte quality and fertilization. Poor oocyte quality\nand subsequent embryonic development could be attributed to mitochondrial dysfunction ( 10 ,  15 ). We hypothesized\nthat vitamin D3 may improve  mtDNA  copy number, mitochondrial membrane integrity, and biogenesis.\nIn the present study, we demonstrated that mitochondrial biogenesis could be upregulated after 24 hours of treatment with vitamin D3. The findings showed that vitamin\nD3, as a supplementation, improves the main mitochondrial biogenesis marker ( TFAM ) in the granulosa cells of\nPCOS ovaries. According to some evidence,  TFAM  plays\nan important role in mitochondrial biogenesis ( 12 ,  30 - 33 ).\nIt is revealed that total antioxidant capacity (TAC) raise\nby vitamin D3 and also vitamin D3 may alleviate the hormonal disturbances in women with PCOS ( 34 ,  35 ). Since\nour results showed that vitamin D3 has an improvement\neffect on ovulation problems and follicular disruption, we\ncould understand that vitamin D3 might have an important role in declining the atretic follicles and alleviating\nthe development of follicles via upregulating the mitochondrial biogenesis main gene and mitochondrial membrane integrity.\nIn this study, for the first time, we have shown the vitamin D3 effect on  mtDNA  copy number and mitochondrial\nmembrane integrity in a mouse model of PCOS granulosa\ncells. Our results revealed that most of the mitochondria\nin the PCOS group were spherical with almost no cristae.\nIn line with our study, Longfei et al. revealed a distorted\nmitochondrial structure and diminished membrane integrity in the PCOS group ( 10 ). Oocytes of the PCOS mouse\nmodel, induced with DHEA, have demonstrated disrupted\nmitochondrial biogenesis, decreased  mtDNA  copy number, and distorted mitochondrial ultrastructure that is in\nagreement with our findings in this study ( 10 ,  36 ). Ding\net al. also observed that mitochondrial dysfunction, due\nto  mtDNA  mutation, has a role in the manifestation of\nPCOS symptoms that is in line with our results ( 37 ). Reduced  mtDNA  copy number is associated with poor oocyte quality and subsequent compromised embryo development and implantation ( 10 ) ( 15 ). Our findings showed\ndecreased  mtDNA  copy number in the PCOS group (nontreated), which is increased upon treatment with vitamin\nD3\nIn line with this finding, researchers showed reduced\nmtDNA copy number in the PCOS patients ( 37 ,  38 ).\nAlso, in agreement with our findings, a case-control study\nshowed reduced  mtDNA  copy number in Korean women\nsuffering from PCOS ( 9 ,  10 ,  39 ). Bhanoori et al. ( 9 ) also\ndemonstrated that mtDNA copy number severely decreased in PCOS patients.\n\nAccording to our results, mtDNA copy number, the\nbiogenesis might be affected by vitamin D3 in PCOS\ngranulosa cells. We nominate that mitochondrial biogenesis genes expression might be increased by vitamin\nD3. Therefore, vitamin D3 can have a significant role in\nthe alleviation of mitochondria and follicular damages in\nPCOS ovaries. However, extensive studies are needed to\ndetermine the optimal dose and duration of treatment with\nvitamin D3 in PCOS women.","source_license":"CC-BY-4.0","license_restricted":false}